Name: the preparation of drosophila embryos for live-imaging using the hanging drop protocol
Uploaded: 2009-03-13T00:00:00
Description: Watch this Scientific Journal Video about The Preparation of Drosophila Embryos for Live-Imaging Using the Hanging Drop Protocol at JoVE.com

We gratefully acknowledge support to B.H.R. through a Discovery Grant as well as a Research Tools and Instrument Grant from the Natural Sciences and Engineering research Council of Canada (NSERC). We also acknowledge H. Oda and the Bloomington Drosophila stock Center for providing genetic stocks that were used in the example live imaging sequences.

Preparation
<ol>
<li>Collect embryos on standard grape-juice agar plates4. It is convenient to use an automated Drosophila Egg Collector (Flymax Scientific Equipment Ltd.). Using synchronous staged embryo collections will reduce the number of dechorionations that must be performed in order to obtain adequate numbers of embryos at the desired developmental stage.</li>
<li>Prepare a dechorionation slide by attaching a piece of double-sided tape to a microscope slide; remove the backing from the tape.</li>
<li...

<ol>
	<li>Kiehart, D. P., Galbraith, C. G., Edwards, K. A., Rickoll, W. L., Montague, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multiple+forces+contribute+to+cell+sheet+morphogenesis+for+dorsal+closure+in+Drosophila.">Multiple forces contribute to cell sheet morphogenesis for dorsal closure in Drosophila.</a> J. Cell Biol. 149, 471-490 (2000).</li><li>Schock, F., Perrimon, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cellular+processes+associated+with+germ+band+retraction+in.">Cellular processes associated with germ band retraction in.</a> , 248-2429 (2002).</li><li>Reed, B. H., Wilk, R., Schock, F., Lipshitz, H. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Integrin-dependent+apposition+of+Drosophila+extraembryonic+membranes+promotes+morphogenesis+and+prevents+anoikis.">Integrin-dependent apposition of Drosophila extraembryonic membranes promotes morphogenesis and prevents anoikis.</a> Curr. Biol. 14, 372-380 (2004).</li><li>Wieschaus, E. p., Nusslein-Volhard, C., Roberts, D. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Drosophila: a practical approach. , 199-227 (1986).</li></ol>

We describe a new method of preparing Drosophila embryos for live imaging analysis, which we call the hanging drop protocol. Unfortunately, it is not possible to use the hanging drop protocol if working with an inverted microscope. In this case the sandwiching technique (as described in the abstract above) must be used and compression of the embryos remains a concern.
In experiments where cell shape and size are measured in order to calculate forces associated with morphogenetic movement, the...

Standard equipment and materials required to prepare embryos for live-imaging using the hanging drop protocol include the following items:  microscope slides, coverslips (22 x 40 mm, No. 2), double-sided tape, jeweller's forceps (Dumont style No. 5), halocarbon oil series 56 and series 700 (Halocarbon Products Corp.), a utility knife or single edge razor blade, tissue paper, scissors, distilled water, general purpose tape, and a pipet suitable for delivering 20-40 &#956;l.  The live imaging protocol also requires a custom-made live imaging chamber.  This is prepared by cutting a 5 mm think polycarbonate plastic sheet to the dimensions of a standard microscope slide (75 X 25 mm) and using a rotor to create a 3mm deep depression in the slide (20 X 55 mm).  Access to a stereomicroscope (dissecting microscope) and a fiber-optic illumination source is also required.  

the preparation of drosophila embryos for live-imaging using the hanging drop protocol

Green fluorescent protein (GFP)-based timelapse live-imaging is a powerful technique for studying the genetic regulation of dynamic processes such as tissue morphogenesis, cell-cell adhesion, or cell death. Drosophila embryos expressing GFP are readily imaged using either stereoscopic or confocal microscopy. A goal of any live-imaging protocol is to minimize detrimental effects such as dehydration and hypoxia. Previous protocols for preparing Drosophila embryos for live-imaging analysis have involved placing dechorionated embryos in halocarbon oil and sandwiching them between a halocarbon gas-permeable membrane and a coverslip1-3. The introduction of compression through mounting embryos in this manner represents an undesirable complication for any biomechanical-based analysis of morphogenesis. Our method, which we call the hanging drop protocol, results in excellent viability of embryos during live imaging and does not require that embryos be compressed. Briefly, the hanging drop protocol involves the placement of embryos in a drop of halocarbon oil that is suspended from a coverslip, which is, in turn, fixed in position over a humid chamber. In addition to providing gas exchange and preventing dehydration, this arrangement takes advantage of the buoyancy of embryos in halocarbon oil to prevent them from drifting out of position during timelapse acquisition. This video describes in detail how to collect and prepare Drosophila embryos for live imaging using the hanging drop protocol. This protocol is suitable for imaging dechorionated embryos using stereomicroscopy or any upright compound fluorescence microscope.

Watch this Scientific Journal Video about The Preparation of Drosophila Embryos for Live-Imaging Using the Hanging Drop Protocol at JoVE.com

The Preparation of Drosophila Embryos for Live-Imaging Using the Hanging Drop Protocol

A simple, inexpensive, and effective method of preparing Drosophila embryos for live-imaging analysis is presented. Our protocol provides humidity and gas exchange and does not compress the Drosophila embryo. This method is suitable for GFP-based live imaging of Drosophila embryos using a stereomicroscope or upright compound microscope.

The Preparation of Drosophila Embryos for Live-Imaging Using the Hanging Drop Protocol

Green fluorescent protein (GFP)-based timelapse live-imaging is a powerful technique for studying the genetic regulation of dynamic processes such as ...

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